The Role Of MiRNA/RNF8 Axis In Cancer Metastasis

Cancer is a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body.Invasion and metastasis are the main causes of mortality in cancer patients.Epithelial-mesenchymal transition(EMT)plays an important role in the metastasis of cancer cells.RNF8,a RING finger E3 ligase that has been best characterized for its involvement in DNA damage repair and sperm formation via ubiquitination,has been reported to promote metastasis in breast cancer and hepatocellular carcinoma recently.However,the role of RNF8 in other type of cancers were not well documented.Meanwhile,the upstream regulatory factors of RNF8 in cancer are rarely reported.In this thesis,we sought to study the effect of RNF8 in lung cancer,a worldwide malignant tumor with the highest lethality,and further explored the potential micro RNAs(miRNAs)which could target RNF8,as well as the involvement of miRNA-RNF8 axis in cancer metastasis.First,our studies showed that RNF8 was highly expressed in lung cancer tissues and had a negative correlation with Overall Survival of patients.RNF8 overexpression promoted the EMT process and migration ability of lung cancer cells,whereas RNF8knock-down showed the opposite effect.RNF8 overexpression activated PI3K/Akt and ERK signaling pathways,while knockdown of RNF8 inactivated Akt and MAPK activity and reduced the expression of Snail,Zeb1 and Slug.To further reveal the signaling pathway by which RNF8 was involved lung cancer metastasis,immunoprecipitation was performed with RNF8 overexpression cells.Results showed that RNF8 can directly intact with Slug and MAPK1,and RNF8 promotes the K63-Ub of Slug.Furthermore,knockdown of Slug can abolish RNF8 dependent EMT of A549 cells,and overexpression Slug can rescue RNF8 dependent H1299 cells MET.These results show that RNF8 contributes to lung tumorigenesis by promoting EMT.RNF8 might be a potential therapeutic target for lung cancer.MiRNAs represent a class of endogenous non-coding genes that regulate gene expression in post-transcription level.In recent decades,miRNAs have been reported to regulate many important biological processes such as epithelial�mesenchymal transition(EMT),which is recognized as a key procedure of tumor metastasis.Therefore,in order to explore the upstream regulation mechanism of RNF8,we also investigated the role of miRNA targeting RNF8 in breast cancer.MiRNAs which could potentially target RNF8 m RNA were predicted using five online databases,and results showed that the 3'UTR region of RNF8 has multiple potential binding site of mir-214 and mir-622,which suggested that RNF8 could be the direct target of these two miRNAs.We found that miR-214 expression was significantly increased in breast cancer tissues compared with adjacent non-cancerous tissues.Overexpression of miR-214 inhibits cell proliferation and invasion of breast cancer cell,while suppression of miR-214 by chemically modified antagomir enhances proliferation and migration of breast cancer cells.We also verified miR-214 could modulate EMT process via downregulating RNF8 and the EMT process induced by down-reglation of miR-214 co?Ld be reversed by knockdown of RNF8.At the same time,we also found for the first time that miR-622 inhibits EMT in breast cancer cells.MiR-622 can regulate EMT process by downregulating RNF8,while knockdown of RNF8 can reverse the the EMT process induced by down-regulation of miR-622.These results indicated a negative link between miR-622 / miR-214 and RNF8 both in expression level and the role of the EMT process,and further confirmed that RNF8 is a direct target of miR-622 / miR-214,suggesting that artificially increasing the expression of miR-622 / miR-214 could be a potential therapeutic strategy for breast cancer treatment.Taken together,our results have revealed the role of miR-622/ miR-214-RNF8 axis in cancer metastasis and suggest that the manipulation of this pathway could be a useful strategy to retard cancer metastasis.